Ouabain is a specific inhibitor of mammalian Na+, K+ -ATPase (NKA) and it has, along with the other cardiac glycosdies, a pronounced inotropic effect on heart muscle. The cardiac glycosides are still the most widely used medication for the treatment of heart failure but their pharmacologic action is not well understood. The concept has developed that their inotropic effect may be the result of an inhibitory action on NKA. The elucidation of the molecular interaction of ouabain with NKA would provide information on the structure, function, and mode of action of this important enzyme. We have isolated mutant BALB/3T3 cells following MNNG mutagenesis that are resistant to 2-5mM ouabain, 10 times the concentration sufficient to kill wild-type cells. We have also successfully transferred the resistant phenotype to NIH/3T3 cells by transformation with a CaPO4 precipitate of resistant BALB DNA. These two features of the system; detectability and transfer of the gene are two prerequisites that have been met and will now allow us to pursue the cloning of the gene conferring ouabain resistance. Studies are proposed that will allow us to identify the protein coded for by this gene; predictably a subunit or subunits of NKA, to determine its amino acid sequence, and by comparison with the protein from normal cells to identify the site of the mutation leading to resistance. Our studies will also characterize the structural organization of the gene. Specifically we propose to 1) clone the gene by using cell transformation, recombinant DNA, and gene rescue techniques, 2) characterize the structure of the cloned gene, 3) to purify hybridizable mRNA from mouse kidney, translate it in vitro and characterize the product, 4) prepare cDNA clones from the mRNA and determine the nucleotide sequence and from that a deduced amino acid sequence, 5) to determine by R-looping the number and location of any intervening regions in the cloned gene, 6) to use the cDNA clone to probe a normal mouse library and determine the number of copies of the gene, 7) if there is reiteration of the gene to determine if their developmental regulation of expression might account for differences in NKA sensitivity to ouabain observed in different tissues or at different stages of development.